1. Field of the Invention
The invention relates generally to growth differentiation factor (GDF) receptors, and more specifically to GDF-8 (myostatin) receptors, to compositions that affect myostatin signal transduction in a cell, and to methods of using such compositions to modulate myostatin signal transduction in a cell.
2. Background Information
The amount of time, effort and money spent in the United States each year by individuals intent on losing weight is staggering. For many of these individuals, the goal is not merely to look better, but more importantly to avoid the inevitable medical problems associated with being overweight.
Greater than half of the adult population in the United States is considered to be overweight. Furthermore, twenty to thirty percent of adult men and thirty to forty percent of adult women in the United States are considered obese, with the highest rates occurring among the poor and minorities. Obesity, which is defined a being at least about twenty percent above the mean level of adiposity, has dramatically increased in prevalence over the past few decades and is becoming a major problem among the pediatric population. Twenty percent of all children are now considered overweight, a number that represents a doubling over the past five years.
Obesity and the medical problems directly attributable to it are a major cause of morbidity and mortality throughout the world. Obesity is a major risk factor for the development of various pathologic conditions, including atherosclerosis, hypertension, heart attack, type II diabetes, gallbladder disease, and certain cancers, and contributes to premature death. Heart disease is the leading cause of mortality in the United States, and type II diabetes afflicts over 16 million people in the United States and is one of the leading causes of death by disease.
More than eighty percent of type II diabetes occurs in obese persons. Although type II diabetes affects all races, it is particularly prevalent among Native Americans, African Americans and Hispanics. Significantly, type II diabetes, which used to occur almost exclusively in adults over age forty, now occurs in children, with reported cases having almost tripled over the last five years. Type II diabetes, also called non-insulin dependent diabetes, is characterized by reduced secretion of insulin in response to glucose and by resistance of the body to the action of insulin, even though insulin levels in the circulation generally are normal or elevated. Type II diabetes affects the function of a variety of different tissues and organs and can lead to vascular disease, renal failure, retinopathy and neuropathy.
In contrast to the medical problems associated with obesity, the severe weight loss that commonly occurs in patients with certain chronic diseases also presents a challenge to medical intervention. The molecular basis for this weight loss, referred to as cachexia, is not well understood. It is clear, however, that cachexia complicates management of such diseases and is associated with a poor prognosis for the patients. The effects of cachexia are evident in the wasting syndrome that occurs in cancer and AIDS patients.
Although great efforts have been made in attempting to elucidate the biological processes involved in regulating body weight, the results have provided more fanfare than actual value. For example, the discovery of leptin has been hailed as a breakthrough in understanding the molecular basis for fat accumulation in humans, and, with it, the promise of a cure for obesity. Studies in animals indicated that leptin is involved in transmitting internal signals regulating appetite, and suggested leptin could be useful for treating humans suffering from obesity. Progress in using leptin for treating obesity has been slow, however, and, thus far, leptin has not met initial expectations.
Treatment of the morbidly obese currently is limited to surgery to remove portions of the intestine, thereby reducing the amount of food (and calories) absorbed. For the moderately obese, the only xe2x80x9ctreatmentxe2x80x9d is eating a healthy diet and exercising regularly, a method that has proved modestly successful at best. Thus, a need exists to identify the biological factors involved in regulating body weight, including muscle development and fat accumulation, such that methods for treating disorders such as obesity and cachexia can be developed. The present invention satisfies this need and provides additional advantages.
The present invention relates to a substantially purified GDF receptor. A GDF receptor of the invention can be, for example, a myostatin receptor, a GDF-11 receptor, or other GDF receptor. A myostatin receptor, for example, interacts specifically at least with myostatin, and also can interact specifically with one or a few additional mature GDF peptides as well. Polynucleotides encoding a GDF receptor, antibodies that specifically interact with a GDF receptor, and the like also are provided.
The present invention also relates to a method of modulating an effect of a GDF by affecting signal transduction effected by the GDF. By way of example, a method of modulating an effect of myostatin on a cell by contacting the cell with an agent that affects myostatin signal transduction in the cell is provided. In one embodiment, the agent alters a specific interaction of myostatin with a myostatin receptor expressed by the cell, thereby modulating myostatin signal transduction in the cell. The myostatin receptor can be an activin receptor, or can be any other receptor that can be contacted by a mature myostatin or functional peptide portion thereof such that myostatin signal transduction is activated. In another embodiment, the agent binds to a myostatin receptor, thereby enhancing myostatin binding to the receptor or competing with myostatin for the receptor. As such, the agent can increase myostatin signal transduction, or can reduce or inhibit myostatin signal transduction. In still another embodiment, the agent acts intracellularly to alter myostatin signal transduction in the cell.
An agent useful for modulating GDF signal transduction in a cell can be a peptide, a peptidomimetic, a polynucleotide, a small organic molecule, or any other agent, and can act as an agonist of GDF signal transduction or as an antagonist of GDF signal transduction. In one embodiment, the peptide agent alters a specific interaction of myostatin with a myostatin receptor. Such a peptide agent can be, for example, a peptide that binds or otherwise sequesters myostatin, thereby affecting the ability of myostatin to interact specifically with its receptor. Such agents are exemplified by a mutant myostatin receptor, for example, a soluble extracellular domain of a myostatin receptor, which can specifically interact with myostatin; by a myostatin prodomain, which can specifically interact with myostatin; and by a mutant myostatin polypeptide that is resistant to proteolytic cleavage into a prodomain and mature myostatin and can interact specifically with myostatin, and are useful as myostatin signal transduction antagonists, which reduce or inhibit myostatin signal transduction in a cell.
In another embodiment, the peptide agent can specifically interact with a myostatin receptor expressed by a cell, thereby competing with myostatin for the receptor. Such a peptide agent is exemplified by an anti-myostatin receptor antibody or by an anti-idiotypic antibody of an anti-myostatin antibody. Such a peptide agent provides the additional advantage that it can be selected not only for its ability to interact specifically with a myostatin receptor, thereby competing with myostatin for the receptor, but can be further selected to have an ability to not activate or not activate myostatin signal transduction. Thus, a peptide agent that specifically interacts with a myostatin receptor expressed by a cell, and activates myostatin dependent signal transduction can be used as a myostatin agonist to increase myostatin signal transduction in the cell, whereas a peptide agent that specifically interacts with a myostatin receptor expressed by a cell, but does not activate myostatin signal transduction can be used as a myostatin antagonist to reduce or inhibit myostatin signal transduction in the cell.
An agent useful in a method of the invention also can be a polynucleotide. Generally, but not necessarily, the polynucleotide is introduced into the cell, where it effects its finction either directly, or following transcription or translation or both. For example, the polynucleotide agent can encode a peptide, which is expressed in the cell and modulates myostatin activity. Such an expressed peptide can be, for example, a mutant myostatin receptor such as a soluble myostatin receptor extracellular domain; a myostatin receptor extracellular domain operatively associated with a membrane anchoring domain; or a mutant myostatin receptor lacking protein kinase activity.
A peptide expressed from a polynucleotide agent also can be a peptide that affects the level or activity of an intracellular polypeptide component of a GDF signal transduction pathway. The intracellular polypeptide can be, for example, an Smad polypeptide such as a dominant negative Smad, which, as disclosed herein, can affect myostatin signal transduction in a cell. Thus, a polynucleotide agent can encode a dominant negative Smad 2, Smad 3 or Smad 4 polypeptide, which, upon expression in the cell, reduces or inhibits myostatin signal transduction in the cell; or can encode a Smad 6 or Smad 7 polypeptide, which, upon expression, decreases myostatin signal transduction in the cell. A polynucleotide agent also can encode an intracellular c-ski polypeptide, the expression of which can reduce or inhibit myostatin signal transduction.
A polynucleotide agent useful in a method of the invention also can be, or can encode, an antisense molecule, a ribozyme or a triplexing agent. For example, the polynucleotide can be (or can encode) an antisense nucleotide sequence such as an antisense c-ski nucleotide sequence, which can increase myostatin signal transduction in a cell; or an antisense Smad nucleotide sequence, which can increase myostatin signal transduction or can reduce or inhibit myostatin signal transduction, depending on the particular Smad antisense nucleotide sequence.
The present invention also relates to a method of ameliorating the severity of a pathologic condition, which is characterized, at least in part, by an abnormal amount, development or metabolic activity of muscle or adipose tissue in a subject. Such a method encompasses modulating GDF signal transduction in a cell associated with the pathologic condition, for example, modulating myostatin signal transduction in a muscle cell or an adipose tissue cell in the subject. Various pathologic conditions are amenable to amelioration using a method of the invention, including, for example, wasting disorders such as cachexia, anorexia, muscular dystrophies, neuromuscular diseases; and metabolic disorders such as obesity and type II diabetes.
The present invention further relates to a method of modulating the growth of muscle tissue or adipose tissue in a eukaryotic organism by administering to the organism an agent that affects signal transduction mediated by a GDF receptor. In one embodiment, a method of modulating the growth of muscle tissue or adipose tissue is performed by administering an agent that affects myostatin signal transduction. In another embodiment, the agent affects GDF-11 signal transduction, or myostatin and GDF-11 signal transduction. The agent can be, for example, an agent alters the specific interaction of myostatin with a myostatin receptor, an agent that reduces or inhibits the specific interaction of myostatin with a myostatin receptor, or any other agent as disclosed herein. The eukaryotic organism can be a vertebrate, for example, mammalian, avian or piscine organism, or can be an invertebrate, for example, a mollusk such as a shrimp, a scallop, a squid, an octopus, a snail, or a slug.
The present invention also relates to a method of identifying an agent that specifically interacts with a growth differentiation factor (GDF) receptor. Such a screening assay of the invention can be performed, for example, by contacting a GDF receptor with a test agent, and determining that the test agent specifically interacts with the GDF receptor, thereby identifying an agent that specifically interacts with a GDF receptor. The GDF receptor can be any GDF receptor, particularly a myostatin receptor, and the agent can be a GDF receptor agonist, which increases GDF signal transduction, or a GDF receptor antagonist, which reduces or inhibits GDF signal transduction. Such a method of the invention is useful for screening a library of test agents, particularly a combinatorial library of test agents.
The present invention also provides a virtual representation of a GDF receptor or a functional peptide portion of a GDF receptor, for example, a virtual representation of GDF 8 receptor or GDF-11 receptor. In one embodiment, the virtual representation includes an agent that interacts specifically with the GDF receptor. As such, the invention further provides a method of identifying an agent that interacts specifically with a growth differentiation factor (GDF) receptor or a functional peptide portion of a GDF receptor by using a computer system. For example, the method can be performed by testing a virtual test agent for the ability to interact specifically with a virtual GDF receptor or functional peptide portion thereof; and detecting a specific interaction of the virtual test agent with the virtual GDF receptor or functional peptide portion thereof, thereby identifying an agent that interacts specifically with a GDF receptor or functional peptide portion thereof.